The overall aim of this proposal is to study the developmental biology of a major arm of the mucosal immune system, namely, intraepithelial lymphocytes (IEL). There are three major goals of our studies. 1) To study the mechanism of selection of V64+ murine IEL. Analysis of T cell receptor (Tcr) junctional diversity and the relative contribution of the two V-delta-64 genes will be performed. Recombinant inbred and backcross mice will be used to map the element(s) involved in the selection process. This system offers an excellent opportunity to analyze the in vivo mechanism of gamma delta T cell selection and will lead to a better understanding of gamma delta T cell specificity and function. 2)To study the differentiation pathway of IEL. The intestine may be a site of extrathymic T cell development. If true, precursors of IEL, and perhaps other T cells, may reside in the intestinal mucosa. Reconstitution of severe combined immunodeficient mice (SCID) using IEL and thymocyte subsets will be performed in order to address this possibility. Additionally, recombination activating gene (RAG) expression will be determined in IEL subsets as a further measure of maturational stage. A second site of T cell maturation may be important for organ-specific immunity and could be a potential source of autoimmune reactivity. 3)To determine Tcr V region, usage and function in IEL subsets. "Forbidden" alpha-beta Tcrs are expressed on a subset of IELs but a complete analysis of V region usage in all IEL subsets has not been performed. Fluorescence flow cytometry will be used to analyze V region expression of IEL subsets including CD4+8+ IELs. Most importantly, the functional qualities of non-deleted IEL will be tested. In conjunction with the reconstitution experiments the influence of the thymus on the expression of "forbidden" Tcrs will be assessed. Considering the potential importance of the mucosal immune system in the etiology of intestinal autoimmune disorders and in combatting infectious diseases it is imperative to obtain a detailed understanding of the control of mucosal T cell development and function. The experiments proposed will significantly further our knowledge in this area.